Biomedical subjects

L Overton

Publications and source records attributed to L Overton.

8 recordsLinked to original sources

PubMed2001

Mouse BAC ends quality assessment and sequence analyses.

A large-scale BAC end-sequencing project at The Institute for Genomic Research (TIGR) has generated one of the most extensive sets of sequence markers for the mouse genome to date. With a sequencing success rate of >80%, an average read length of 485 bp, and ABI3700 capillary sequencers, we have generated 449,234 nonredundant mouse BAC end sequences (mBESs) with 218 Mb total from 257,318 clones from libraries RPCI-23 and RPCI-24, representing 15x clone coverage, 7% sequence coverage, and a marker every 7 kb across the genome. A total of 191,916 BACs have sequences from both ends providing 12x genome coverage. The average Q20 length is 406 bp and 84% of the bases have phred quality scores > or = 20. RPCI-24 mBESs have more Q20 bases and longer reads on average than RPCI-23 sequences. ABI3700 sequencers and the sample tracking system ensure that > 95% of mBESs are associated with the right clone identifiers. We have found that a significant fraction of mBESs contains L1 repeats and approximately 48% of the clones have both ends with > or = 100 bp contiguous unique Q20 bases. About 3% mBESs match ESTs and > 70% of matches were conserved between the mouse and the human or the rat. Approximately 0.1% mBESs contain STSs. About 0.2% mBESs match human finished sequences and > 70% of these sequences have EST hits. The analyses indicate that our high-quality mouse BAC end sequences will be a valuable resource to the community.

Animals↗

PubMed1999

A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors.

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.

Amino Acid Sequence↗

PubMed1997

Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system.

Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.

Animals↗

PubMed1997

Detailed characterization of a purified type 4 phosphodiesterase, HSPDE4B2B: differentiation of high- and low-affinity (R)-rolipram binding.

We have overexpressed in a baculovirus expression system, and purified to > 95% homogeneity, milligram quantities of a human recombinant rolipram-sensitive cAMP phosphodiesterase, HSPDE4B2B (amino acid residues 81-564). The protein expression levels were approximately 8 mg of HSPDE4B2B (81-564) per liter of Sf9 cells. The Km of the purified enzyme for cAMP was 4 microM and the Ki for the Type 4 phosphodiesterase-specific inhibitor (R)-rolipram was 0.6 microM. The specific activity of the purified protein was 40 mumol/min/mg protein. A nonequilibrium filter binding assay revealed a high-affinity (R)-rolipram binding site on the purified enzyme with a Kd of 1.5 nM and a stoichiometry of 0.05-0.3 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Equilibrium dialysis experiments revealed a single binding constant of 140 nM with a stoichiometry of 0.75 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Size exclusion chromatography and analytical ultracentrifugation experiments suggest that the protein exists in multiple association states larger than a monomer. Proteolysis experiments revealed a 43-kDa fragment that contained catalytic and rolipram-inhibitable activities, but the fragment showed no high-affinity (R)-rolipram binding. Based on the proteolytic cleavage studies a 43-kDa protein was constructed, expressed, and purified. This protein, HSPDE4B2B (152-528), had Km and Vmax similar to those of the HSPDE4B2B (81-564) protein, but did not exhibit high-affinity (R)-rolipram binding. The protein did show low-affinity (R)-rolipram binding using the equilibrium binding assay. These results show that a low-affinity binding site for (R)-rolipram is solely contained within the catalytic domain of HSPDE4B2B, whereas high-affinity (R)-rolipram binding requires residues within the catalytic domain and residues flanking N- and/or C-terminal to the catalytic region.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1996

Comparative analyses of relative ERCC3 and ERCC6 mRNA levels in gliomas and adjacent non-neoplastic brain.

Nucleotide excision repair (NER) is an ordered process in nonmalignant cells, in both human and nonhuman systems. We previously reported that in human brain there is discordant mRNA expression of excision repair cross-complementing (ERCC) 1 and ERCC2 in malignant tissues, concurrent with excellent concordance of these genes in nonmalignant tissues from the same patients. Here we have extended these studies to compare low-grade tumors to high-grade tumors and to include ERCC3 (which links DNA repair with DNA transcription) and ERCC6 (which is essential for gene-specific repair). Glial tumor and adjacent normal brain specimens from 19 individuals were studied. Paired malignant and nonmalignant tissues were obtained from 12 of these patients. For ERCC3, there was excellent concordance of mRNA expression between malignant and nonmalignant tissues from the same individuals (P = 0.003). For ERCC6, no concordance was observed (P = 0.314). Tumor tissue from patients with high-grade gliomas exhibited marked discordance of mRNA expression patterns in situations in which good concordance was observed in tumor tissue from low-grade gliomas. We previously established that malignant brain tumors show increased disorder of genes in the NER process, as compared with nonmalignant tissues. These data suggest that increasing disorder in the NER process may occur as cells move from low-grade to high-grade malignancy.

Astrocytoma↗

PubMed1994

Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor.

The genetic loci agouti and extension control the relative amounts of eumelanin (brown-black) and phaeomelanin (yellow-red) pigments in mammals: extension encodes the receptor for melanocyte-stimulating hormone (MSH) and agouti encodes a novel 131-amino-acid protein containing a signal sequence. Agouti, which is produced in the hair follicle, acts on follicular melanocytes to inhibit alpha-MSH-induced eumelanin production, resulting in the subterminal band of phaeomelanin often visible in mammalian fur. Here we use partially purified agouti protein to demonstrate that agouti is a high-affinity antagonist of the MSH receptor and blocks alpha-MSH stimulation of adenylyl cyclase, the effector through which alpha-MSH induces eumelanin synthesis. Agouti was also found to be an antagonist of the melanocortin-4 receptor, a related MSH-binding receptor. Consequently, the obesity caused by ectopic expression of agouti in the lethal yellow (Ay) mouse may be due to the inhibition of melanocortin receptor(s) outside the hair follicle.

Adenylyl Cyclases↗

PubMed1994

Recombinant protein expression in a Drosophila cell line: comparison with the baculovirus system.

In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinant Drosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by the Drosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [3H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.

Animals↗

PubMed1993

High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc.

The mammalian enzyme protein farnesyltransferase is a heterodimeric protein that catalyzes the addition of a farnesyl isoprenoid to a cysteine in ras proteins. Since oncogenic forms of ras proteins require the farnesyl group for transforming activity, the structure and mechanism of this enzyme are important to define. However, such studies have been difficult to approach because of the low abundance of the enzyme in mammalian tissues and hence the problems of obtaining large quantities of the protein. We report here the co-expression of the two subunits of protein farnesyltransferase by Sf9 cells infected with a recombinant baculovirus containing the coding sequences of both polypeptides. This results in the production of milligram quantities of enzyme which can be readily purified by conventional chromatographic methods. The individual subunits of the enzyme can also be expressed in the Sf9 cells, but the ability to reconstitute active enzyme from extracts containing individual subunits is quite low. In contrast, the enzyme produced by co-expression of the two subunits is fully active and retains the properties of the mammalian form, including the specificity for the COOH-terminal amino acid of substrate proteins and the ability to bind short peptides encompassing the prenylation site of a ras protein. Furthermore, through atomic absorption analysis of the purified protein, we have confirmed the previous tentative assignment of protein farnesyltransferase as a zinc metalloenzyme by demonstrating that it contains an essentially stoichiometric amount of zinc. The ability to produce and purify milligram quantities of protein farnesyltransferase readily will allow detailed mechanistic and structural studies on this enzyme.

Alkyl and Aryl Transferases↗